The goals of this proposal are to study the structure and biosynthesis of prenylated proteins. Prenylation is a recently discovered posttranslational modification of proteins. These proteins are derivatized with polyisoprenoids, molecules normally associated with sterol, dolichol, and coenzyme Q synthesis. There are approximately a dozen prenylated proteins, two of which have been identified. One, a Ras protein, is functionally activated by prenylation, indicating a role for in the transformation of cells, a cancer-related event. The other, Lamin B, is a structural protein of the nuclear membrane. Since proteins of such diverse function are prenylated, a general function for this modification is indicated. We have identified two prenyl groups, differing in molecular weight. Both are linked as thioethers to cysteine, which is carboxy terminal. The smaller is a diterpene (C20). The larger has not been fully characterized. The primary goal is to determine unequivocally the structures of these isoprenoids. Using the prenyl group as a guide, various prenylated proteins will be purified. After proteolytic cleavage, the prenyl-bearing residue will be isolated for amino acid sequencing. These sequences will help to identify these proteins for amino acid sequencing. These sequences will help to identify these proteins. The prenyl residue is a unique substituent and will provide a route for selective labeling and manipulation of these proteins. Ozonolysis will be used to cleave their olefinic bonds to aldehydes. Reduction of the aldehydes with [3H]NaBH4 will yield a high specific activity label to assist in isolation and characterization. Alternatively, reaction of the aldehyde with a biotin derivative will yield biotin covalently linked to protein. This will provide a powerful tool for the study of these proteins. Other posttranslational modifications of these proteins are concurrent with prenylation. These include proteolytic removal of a carboxy terminal tripeptide as well as methylation of the carboxyl of the cysteine thus exposed. Our studies will order the sequence of these events. Model peptides will be used to study the prenylation reaction.